Application of bifidobacterium animalis subsp. Lactis BLa80 in preparation of medicine or food for resisting diarrhea caused by rotavirus infection
A technology of animal bifidobacteria and rotavirus, applied in the field of microorganisms, to achieve the effect of preventing and alleviating diarrhea symptoms and excellent anti-rotavirus infection
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preparation example 1
[0043] This preparation example provides a kind of Bifidobacterium animalis subsp.lactis BLa80 bacterial suspension, and its preparation method is as follows:
[0044] Pick a single colony of Bifidobacterium animalissubsp.lactis BLa80 rejuvenated on the plate and inoculate it in 10 mL of TPY liquid medium, culture it anaerobically at 37°C for 12 hours, and inoculate the activated bacterial liquid in 100 mL of 2% inoculum In TPY liquid medium, culture anaerobically at 37°C for 12h, centrifuge at 5000g for 10min at 4°C, take the centrifuged bacterial pellet, resuspend with sterile PBS buffer solution, adjust the concentration of the bacterial suspension to 10 9 cfu / mL to obtain the Bifidobacterium animalis subsp. lactis BLa80 bacterial suspension, and store it at 4°C for future use.
Embodiment 1
[0046] The present embodiment carries out the mensuration of cytotoxicity to the Bifidobacterium animalissubsp.lactis BLa80 bacterial suspension obtained in Preparation Example 1, and the specific steps are as follows:
[0047] Culturing small intestinal epithelial cells: FHs 74Int cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37°C, 5% CO 2 Cultured for 48 hours under the condition of passaging, digested with 0.25% trypsin for about 10 minutes. Specific steps are as follows:
[0048] Attach the cells to the wall, suck off the old culture medium in the culture vessel with a dropper or pipette, and then wash off the residual old culture medium with PBS solution; add 1.5mL digestion solution (0.25% trypsin) to the bottle and gently Shake the culture flask to make the digestive juice flow over the surface of all cells, and then observe under an inverted microscope. When the cells withdraw their protrusions and become rounded or the intercell...
Embodiment 2
[0058] This embodiment carries out virulence assay to rotavirus, with TCID 50 That is, the median tissue culture infectious dose (mediantissue culture infectious dose), refers to the amount of virus that can cause half of the cells in the culture plate or test tube to be pathological or dead, and is used to characterize the titer of rotavirus.
[0059] Propagation of rotavirus: Rhesus macaque kidney cells MA104 were passaged for 3 times, and the cells were inoculated with rotavirus hWa strain after the cells became stable. Take out the rotavirus, dilute the virus solution with 1640 culture medium (without serum), mix the diluted virus with 2 μg / mL trypsin at a ratio of 1:1 (v / v), and incubate at a constant temperature of 37°C for 45 minutes. Add 2 mL of the mixture of cultured rotavirus and trypsin to MA-104 cells, and then incubate at 37°C, 5% CO 2 Incubate in an incubator for 90 minutes. Discard the mixed solution, add 1640 culture medium (without serum) to 8 mL, and add 5...
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